Induction of Gal Transcription from Repressor-Bound DNA (Tryptophan Fluorescence͞derepression͞galactose͞circular Dichroism)

Proc. Natl. Acad. Sci. USA Vol. 94, pp. 2957–2962, April 1997 Biochemistry

Interaction of Gal repressor with inducer and operator: Induction of gal transcription from repressor-bound DNA (tryptophan fluorescence͞derepression͞galactose͞circular dichroism)

SUMANA CHATTERJEE*, YAN-NING ZHOU†,SIDDHARTHA ROY*, AND SANKAR ADHYA†‡

*Department of Biophysics, Bose Institute, Calcutta 700 054, India, and †Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255

Contributed by Sankar Adhya, January 3, 1997

ABSTRACT Gal repressor inhibits transcription from view of the mechanism of induction does not apply to the P1 the gal promoter (P1) when it binds to the cognate operator promoter of the operon. (OE). The repression is relieved by the presence of the inducer The gal operon is transcribed by two promoters, P1 and P2 D-galactose. Compared with its interaction with free repres- (2). Repression of the two gal promoters involves Gal repressor sor, D-galactose binds to the repressor–operator complex with (GalR) binding to two operators, OE and OI (3–6). Derepres- 10-fold reduced affinity as determined by fluorescence en- sion occurs by the interaction of GalR with inducer D-galactose hancement measurements. Thermodynamic analysis and flu- or D-fucose (7). Whereas full repression of both P1 and P2 orescence anisotropy showed that the stability of the repres- requires the histone-like protein HU (8), interaction of re- sor–operator complex is reduced by only 7-fold by the presence pressor with OE alone represses P1, but not P2. GalR⅐OE- of the inducer in the complex. The formation of the inducer– mediated repression of P1 observed in the absence of HU also repressor–operator ternary complex has been confirmed by is relieved by the presence of D-galactose or D-fucose (6). We CD spectral analysis. Fluorescence spectroscopy and energy report here the results of an analysis of (i) formation and transfer experiments suggest that individual allosteric effects stability of various binary and ternary complexes of operator, of the two ligands, inducer and operator, on Gal repressor are repressor, and inducer by fluorescence and CD spectroscopy, responsible for the slightly weakened stability of the ternary (ii) the antagonistic effect of inducer or operator on the complex compared with the stability of the inducer–repressor binding of the other to repressor, and (iii) DNA-induced and repressor–operator complexes. In vitro transcription re- conformational changes in GalR. These results lead us to sults demonstrated full derepression of transcription of the P1 propose that inducer derepresses transcription in this system promoter under conditions in which the concentrations of the without dissociating the repressor from the operator. inducer–repressor binary complex are severalfold higher than the dissociation constant of the inducer–repressor–operator MATERIALS AND METHODS ternary complex into inducer–repressor and free DNA. These results strongly suggest that the inducer binding to the Materials. D-Galactose, D-fucose, adenosine 5Ј-[␥- repressor–operator complex does not lead to dissociation of thio]triphosphate (ATP[␥-S]), and acrylamide were obtained the repressor from the operator during transcription induc- from Sigma. Fluorescein isothiocyanate and 5-({[(2- tion. Because Gal repressor inhibits transcription by modu- iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid (IAEDANS) were purchased from Molecular Probes. Oligo- lating the ␣ subunit of the P1-bound RNA polymerase, we nucleotides purified by HPLC were from Midland Certified conclude that the inducer binding to the operator-bound Reagent (Midland, TX). The sequence of the 20-mer wild-type repressor only allosterically relieves the inhibitory effect of O was 5Ј-TTGTGTAAACGATTCCACTA-3Ј, whereas that repressor on RNA polymerase without dissociating the repres- E of the mutant O was 5Ј-TTGTGTAAATAATTCCACTA-3Ј. sor from DNA. E The sequence of the 29-mer wild-type OE was 5Ј-xACTTCT- TGTGTAAACGATTCCACTAATTT-3Ј, x being a hexyl- In the classical negative control of gene expression, a repressor amino group (2). Polynucleotide kinase was obtained from protein binds to cognate operator element(s) in DNA at or BRL. GalR was purified from a plasmid clone in which the near the promoter and inhibits transcription initiation (1). galR gene was expressed from a late promoter of bacteriophage Induction of transcription can occur in the presence of an T7 as described previously (9). The concentration of GalR inducer molecule that interacts with and causes an allosteric dimer was obtained from its extinction coefficient at 280 nm change in the repressor, which then loses its inhibitory power of 39,600 MϪ1⅐cmϪ1 (10) and is expressed in terms of mono- on transcription. It was originally proposed and commonly mer. Plasmid pSA509 used as the source of DNA in transcrip- believed that repressor sterically prevents RNA polymerase tion assays has been described before (6). from binding to the promoter site. According to this idea, IAEDANS Labeling of OE. Twenty nanomoles of OE DNA because of inducer-generated allosteric change(s), the repres- duplex, 250 ␮mol of ATP[␥-S], and 10 units of T4 polynucle- sor loses its affinity for the operator and dissociates from otide kinase were incubated at 37ЊCfor2hin0.1Mpotassium DNA, thus allowing RNA polymerase to bind. We investigated phosphate buffer, pH 8.0. The mixture was extracted with an the dissociation of repressor from the operator DNA by equal volume of phenol͞chloroform͞isoamyl alcohol inducer under conditions of transcription derepression in the (25:24:1). The aqueous layer was loaded onto a Sephadex G-25 gal operon of Escherichia coli and found that this long-held Abbreviations: IAEDANS, 5-({[(2-iodoacetyl)amino]ethyl}amino)- The publication costs of this article were defrayed in part by page charge naphthalene-1-sulfonic acid; AEDANS, 5-({[(acetyl)amino]ethyl}- payment. This article must therefore be hereby marked ‘‘advertisement’’ in amino)naphthalene-1-sulfonic acid; ATP[␥-S], adenosine 5Ј-[␥-thio] accordance with 18 U.S.C. §1734 solely to indicate this fact. triphosphate. ‡To whom reprint requests should be addressed at: Developmental Copyright ᭧ 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA Genetics Section, Laboratory of Molecular Biology, Building 37, 0027-8424͞97͞942957-6$2.00͞0 Room 2E16, 37 Convent Drive, MSC 4255, Bethesda, MD 20892- PNAS is available online at http:͞͞www.pnas.org. 4255.

2957 Downloaded by guest on October 7, 2021 2958 Biochemistry: Chatterjee et al. Proc. Natl. Acad. Sci. USA 94 (1997)

column (1.5 ϫ 12.5 cm), equilibrated with 0.1 M potassium ␧D and ␧A are the molar extinction coefficients of the donor phosphate (pH 8.0) and eluted. Appropriate fractions were and acceptor, respectively, at the excitation wavelength, and pooled, and IAEDANS was added at a final concentration of CD and CA are the concentrations of the donor and acceptor, 0.5 mM. The mixture was incubated for 90 min in the dark and respectively. The distance between the dansyl group and the extensively dialyzed. The calculated incorporation ratio ob- tryptophan in GalR was determined by the following equation: tained from A260 and A340 was approximately 1.2 mol per mol of oligonucleotide duplex. Ϫ1 1/6 R ϭ Ro(E Ϫ 1) , Synthesis and Purification of Fluorescein-Labeled OE. The 29-mer complementary oligonucleotides with a hexylamino group (x) attached at their 5Ј ends were separately synthesized where R is the distance between the donor and the acceptor, in an Applied Biosystems 381A DNA synthesizer using amino Ro is the distance for which the energy transfer efficiency is link supplied by Applied Biosystems. The oligonucleotides 50%, and E is the experimentally observed energy transfer were deprotected by incubating them at 55ЊC for 24 h in 30% efficiency. ammonium hydroxide, precipitated by 1-butanol, then dried 2 Ϫ4 1/6 3 under vacuum and dissolved in water. Ro ϭ (J␬ Q␩ ) (9.79 ϫ 10 )cm, Ten milligrams of fluorescein 5-isothiocyanate was dissolved in 200 ␮l of 1.0 M NaHCO3͞Na2CO3 buffer (pH 9.0)͞N,NЈ- where J is the overlap integral (a measure of the spectral dimethylformamide water in the ratio of 5:2:3. The dye solu- ͞ overlap), Q is the quantum yield, ␩ is the refraction index, and tion, the oligonucleotide solution, and the carbonate buffer ␬ is the orientation factor. The overlap integral was calculated were mixed in the ratio of 2:5:3, vortexed briefly, and incubated according to the method of Wu and Stryer (12). The value of at 25ЊC for 20 h. The fluorescein-labeled oligonucleotides were ␬2 was taken as 2͞3, which is completely spatially averaged. further purified by C18 reverse-phase HPLC using a linear gradient of 0–60% acetonitrile in 0.1 M triethylammonium Such complete spatial averaging is justified here because of acetate buffer (pH 7.0). The appropriate fractions of each aliphatic linkers separating the 5-({[(acetyl)amino]- labeled oligonucleotide were mixed in equimolar proportion, ethyl}amino)naphthalene-1-sulfonic acid (AEDANS) mole- and then annealed. cule from the DNA as well as the experimentally obtained low Fluorescence. Fluorescence spectra were recorded either in polarization value. The energy transfer experiments were done a Hitachi F3010 or SLM-Aminco (Urbana, IL) spectroflu- in the buffer described above at 25ЊC. The excitation and orometer equipped with computers for spectra addition and emission band passes were 4 nm and 16 nm, respectively. The subtraction facility. Experiments were conducted in a water- emission wavelength was set at 500 nm. circulated thermostat chamber maintained at 25ЊC. Excitation Circular Dichroism. CD spectra were obtained using a Jasco band pass was 1.5 nm, and emission band pass was 20 nm. J-600 spectropolarimeter at 25ЊC. DNA concentration was 2.0 Samples were incubated at 25ЊC for 30 min before conducting ␮M. Ten scans were averaged to produce a CD spectum. The the experiment. This incubation was necessary, because initial bandwidth was 1 nm, and the time constant was 1 s. The dilution of protein from stock solution caused the appearance experiments were conducted in the buffer described above. of slight turbidity, which disappeared within several minutes The buffer spectrum was subtracted from the OE spectrum, upon standing at room temperature. The excitation wave- whereas the buffer-plus-GalR spectrum was subtracted from length was 295 nm; the emission wavelength was 360 nm. In OE-plus-GalR spectrum. The protein did not contribute sig- preliminary experiments, the tryptophan residue in GalR was nificantly to the intensities at the wavelengths used. found to be somewhat photosensitive. The excitation slit was Data Processing. For all titration experiments, control opened only during the fluorescence measurements and only experiments were carried out in which identical volumes of at a single point to reduce exposure time. Repeated measure- buffer were added to the protein solution without the ligand. ments under such conditions yielded constant fluorescence Values from the blank titration were used to correct the values. The experiments were performed in 12.5 mM Tris⅐HCl buffer (pH 8.0) containing 300 mM KCl and 0.5 mM EDTA. volumes for ligand titrations. The corrected fluorescence values then were fitted to an equation of a single class of ligand D-Galactose titrations of GalR were done at 2 ϫ 10Ϫ6 M GalR binding by the nonlinear least-squares method for determining at 25ЊC. For titration of GalR-OE complex by D-galactose, Ϫ6 dissociation constants. The three variable parameters chosen GalR, and OE concentrations were 2.4 ϫ 10 M and 1.2 ϫ Ϫ6 Ϫ6 (initial fluorescence value, fluorescence value at infinite ligand 10 M, respectively. OE titrations were performed at 2 ϫ 10 M GalR. Increasing concentrations of DNA were added to concentration, and the dissociation constant) were systemat- GalR before measuring the emission spectra. The normalized ically varied within a given range. The values that gave the 2 integrated intensity was plotted as a function of OE concen- lowest ␹ were chosen as the best-fit parameters. A similar tration. The fluorescence values were corrected for dilution least-squares fit procedure was used to obtain operator– and inner filter effect. The excitation and emission band passes repressor dissociation constants from anisotropy data. were 2 nm each. In Vitro Transcription. In vitro transcription assays were The direct binding of OE to GalR and to D-galactose⅐GalR done as described previously (9) with slight modifications. The complex was measured fluorometrically by using fluorescein- 348-bp EcoRI–BamHI fragment of pSA509 containing the gal labeled operator DNA in the buffer described above. The promoter region was used as the template. The transcription excitation and emission wavelengths were 490 nm and 525 nm, reaction mixture containing 12.5 mM Tris⅐HCl (pH 8.0), 200 respectively. Excitation and emission band passes were 10 nm. mM potassium chloride, 5 mM magnesium chloride, 2 nM Fluorescence anisotropy was measured after each addition DNA, 20 nM RNA polymerase, 160 or 800 nM GalR, and of GalR as described in Results. different concentrations of D-galactose as indicated in Fig. 7 Fluorescence energy transfer efficiency (E) was calculated was preincubated for 5 min at 37ЊC. The reaction was started from excitation spectra using the following equation (11): by the addition of 0.2 mM ATP, GTP, CTP, and 0.02 mM 32P-labeled UTP. After incubation for 15 min at 37ЊC, the FDϩA͞FA ϭ 1 ϩ (␧DCD͞␧ACA)E, reaction was stopped by the addition of 0.2 vol of 0.2 M EDTA where FDϩA is the fluorescence intensity of the donor– in 40% glycerol with dye. D-Galactose concentrations were 2.0 acceptor pair (dansylated OE and GalR); FA is the fluores- and 30 mM. The RNA was quantified by PhosphorImager 425 cence of the acceptor (dansylated OE) at the same wavelengths; (Molecular Dynamics). Downloaded by guest on October 7, 2021 Biochemistry: Chatterjee et al. Proc. Natl. Acad. Sci. USA 94 (1997) 2959

RESULTS very similar to that observed on binding of D-galactose to free GalR. However, D-galactose bound to the GalR⅐OE complex Inducer Binding to GalR and GalR⅐OE Complex. Binding of Ϫ4 with a dissociation constant (KD3)of2.2ϫ10 M, which is ligands to proteins often causes quenching or enhancement of approximately 10-fold higher than K , the dissociation con- fluorescence of tryptophan, depending on the location of the D1 stant of D-galactose binding to free GalR. This suggests that amino acid(s) in the protein. Such enhancement or quenching although binding of repressor to the operator reduced the can be used to derive binding isotherms. GalR is a dimer affinity of inducer to repressor, because of the sufficient containing only one tryptophan residue per subunit. The residual affinity of the inducer-bound repressor toward the intrinsic fluorescence of this tryptophan was used to monitor operator, the inducer bound to the operator-bound GalR ligand binding. Fig. 1 shows the results of D-galactose, D- without dissociating the complex. fucose, and D-glucose titration of GalR at 1.0 ϫ 10Ϫ6 Mas The inability of D-galactose to dissociate repressor from the monitored by the intrinsic tryptophan fluorescence. Binding of DNA⅐protein complex was further tested by fluorescence D-galactose resulted in a 23% enhancement of tryptophan anisotropy experiments. The O DNA was thiophosphorylated fluorescence, which quickly saturated. No significant fluores- E at the 5Ј end by ATP ␥ S and T4 polynucleotide kinase and cence increase occurred at D-galactose concentrations greater subsequently allowed to react with IAEDANS as described in than 2.0 ϫ 10Ϫ4 M. This enhancement also was accompanied Materials and Methods. The AEDANS-labeled operator at by a small (Ϸ2 nm) red shift of the emission maximum and a 0.5 ϫ 10Ϫ6 M gave an anisotropy value of 0.036 (data not change in acrylamide quenching pattern (data not shown). The shown), indicating significant internal motion of the IAE- data were used to fit a binding equation for a single class of DANS probe. The presence of 1.0 ϫ 10Ϫ6 M GalR monomer binding site using a nonlinear least squares fit procedure. It increased the anisotropy value to 0.064. This value is still low yielded a dissociation constant (K )of2.2Ϯ0.3 ϫ 10Ϫ5 M. D1 compared with that expected for a fully rigid probe, again A similar dissociation constant for the interaction of D- indicating significant internal motion. Such low anisotropy galactose and GalR has been reported previously (13). D- values are not uncommon in 5Ј-end-labeled DNAs because of Fucose is a nonmetabolizable analog of D-galactose and is a flexibility of the linker arm and lack of steric constraints at the slightly weaker inducer (14). Binding of D-fucose also led to an end of the oligonucleotides (15). Further addition of 1.0 ϫ enhancement of tryptophan fluorescence, although the extent Ϫ3 of enhancement (12%) was less, resulting in a dissociation 10 M D-galactose resulted in a very small (less than 5%) decrease in the anisotropy value obtained for GalR plus DNA. constant of 6.0 Ϯ 0.3 ϫ 10Ϫ5 M. D-Glucose, which is not an inducer, caused relatively minor changes in tryptophan fluo- The value in the presence of D-galactose was still much higher rescence. than that of the free operator, indicating that binding of D The binding of D-galactose to operator–repressor complex -galactose to the repressor–operator complex does not lead was studied by monitoring tryptophan fluorescence changes to significant dissociation of the repressor from the operator. D that occurred upon D-galactose binding. The concentration of Determination of the affinity of -galactose to free repres- Ϫ6 Ϫ6 sor and repressor–operator complex allowed an estimation of GalR was 2.4 ϫ 10 M, and that of OE DNA was 1.2 ϫ 10 the relative affinities of OE for the free GalR and the D- M. As will be shown later, Gal repressor bound to OE DNA Ϫ9 galactose–GalR complex based on the binding cycle described with a dissociation constant (KD4)of4ϫ10 M under these in Fig. 2. Two pathways lead from the repressor to the end conditions. Thus, GalR and OE should exist as a 1:1 complex under these conditions. A significant increase of tryptophan product, the inducer–repressor–operator ternary complex. fluorescence was achieved by the addition of increasing con- Thus, centrations of D-galactose, with little enhancement occurring KD1⅐KD2 ϭ KD3⅐KD4 beyond 1.0 ϫ 10Ϫ3 M (Fig. 1). At the high D-galactose concentration, tryptophan fluorescence increased by 23% of or the initial value. The degree of fluorescence enhancement was KD1͞KD3 ϭ KD4͞KD2,

where the KDs are the dissociation constants of the respective binding equations. Thus, the predicted ratio of KD4͞KD2 is 0.1 Ϫ5 as determined from KD1 and KD3 values of 2.2 ϫ 10 M and 2.2 ϫ 10Ϫ4 M, respectively.

FIG. 1. Relative tryptophan fluorescence change (f͞fo) of GalR as a function of hexose concentration. D-Galactose (E), D-fucose (F), D-glucose (Ç), and D-galactose plus 20-mer OE-DNA (Ⅺ). The GalR concentration was 2 ϫ 10Ϫ6 M in all of the experiments, except Ϫ6 Ϫ6 repressor concentration was 2.4 ϫ 10 M and OE DNA was 1.2 ϫ 10 FIG. 2. The binding cycle of GalR (R) to ligands D-galactose (I) M in the titration of GalR⅐OE complex. and operator (O). Downloaded by guest on October 7, 2021 2960 Biochemistry: Chatterjee et al. Proc. Natl. Acad. Sci. USA 94 (1997)

GalR Binding to OE in the Absence and Presence of Inducer. To determine values of KD4 and KD2 experimentally, the affinity of the OE for GalR and D-galactose–GalR complex was determined by fluorescence anisotropy measurements. A 29-bp OE DNA with a hexylamino group at the 5Ј end was labeled with fluorescein isothiocyanate. Fluorescein has a high molar extinction coefficient and quantum yield. The purified duplex was titrated with GalR and anisotropy was measured at each point. A plot of fluorescence anisotropy of the 2.0 ϫ 10Ϫ9 M OE DNA at different GalR concentrations is shown in the absence (Fig. 3A) and in the presence (Fig. 3B)of1ϫ10Ϫ3 M D-galactose. In both cases, fluorescence anisotropy increased by about 40–50%. The observed values of KD4 and KD2 were 4.2 ϫ 10Ϫ9 M and 27.8 ϫ 10Ϫ9 M, respectively. The measured dissociation constant (KD4) of the operator–repressor complex under the present conditions was at least an order of magnitude higher than those obtained by gel electrophoresis and DNase footprinting assays (9, 16). This was expected, as the latter measurements were done in the presence of potassium glutamate instead of KCl and at a lower salt concentration, conditions that increase the affinity of DNA-binding proteins for the target sequences (17). Nevertheless, the binding of D-galactose to GalR reduced the affinity of repressor for the operator by approximately 7-fold. The KD4͞KD2 ratio of 0.15, measured by fluorescence anisotropy, is very similar to the value (0.1) obtained from the indirect binding cycle analysis. The binding of repressor to the operator was further investigated in the presence and absence of inducer by CD spectroscopy. Binding of the GalR to OE leads to a significant change of CD spectra of the operator (10). Fig. 4A shows the CD spectra of the operator, the repressor–operator complex, and the repressor–operator complex in the presence of D- galactose. Binding of GalR to OE at micromolar concentrations, as expected, resulted in a change of the operator CD Ϫ6 spectrum. The direction and magnitude of the spectral change FIG.4. (A) CD spectrum of the operator OE (20-mer) at 2 ϫ 10 was the same as that observed previously (10). The CD spectral M (– – – –), in the presence of GalR at 4.0 ϫ 10Ϫ6 M(⅐⅐⅐⅐), and in the change indicated a DNA distortion that very likely is the DNA presence of GalR at 4.0 ϫ 10Ϫ6 M and D-galactose at 2.0 ϫ 10Ϫ3 M C bending caused by the binding of GalR to O (18). The (——). (B) CD spectrum of the mutant OE operator (20 mer) at 2.0 ϫ E 10Ϫ6 M (– – – –), in the presence of GalR at 4.0 ϫ 10Ϫ6 M (——) and in the presence of GalR at 4.0 ϫ 10Ϫ6 M and D-galactose at 2.0 ϫ 10Ϫ3 M(⅐⅐⅐⅐). The bandwidth was 1.0 nm, and the scan speed was 200 nm͞min. Ten spectra were averaged to improve signal-to-noise ratio.

presence of 2 ϫ 10Ϫ3 M D-galactose did not cause any significant change in the CD spectrum of the repressor– operator complex. Because the D-galactose concentration was 10-fold higher than the apparent dissociation constant of the D-galactose from the operator-repressor complex (Fig. 1), it was evident that the binding of D-galactose to the operator- repressor complex at micromolar concentrations did not lead to the dissociation of repressor from the operator. A control experiment focused on a DNA duplex of the same C size that included a mutant OE sequence (2). Because of a C change of 2 bp, the nature of the spectra of the OE DNA is slightly different from that of Oϩ DNA. Fig. 4B shows the CD spectra of the mutant operator, the mutant operator in the presence of repressor, and the mutant operator in the presence of repressor and D-galactose. The presence of GalR led to only a very small change in the CD spectrum. The addition of D-galactose did not cause any detectable CD change, indicating that the change in the CD spectrum of OE is a consequence of specific binding of GalR. Furthermore, binding of D- galactose to this complex preserved the nature of the complex. OE-Induced Conformational Change in GalR. Similar to the reduction in the affinity of inducer for the operator–repressor FIG. 3. Titration of fluorescein-labeled OE DNA with GalR in the complex compared with that for free repressor, the affinity of absence (A) and in the presence (B) of 1.0 ϫ 10Ϫ3 M D-galactose. Fluorescein-labeled oligonucleotides (29-mer) were prepared as de- the operator for the inducer–repressor complex is lower than Ϫ9 scribed in the text. The OE concentration was 2 ϫ 10 M. Each for free repressor. We tested the possibility that DNA binding reading was time-averaged for 100 sec. The solution conditions are induces an allosteric change in repressor conformation by described in Materials and Methods. measuring the tryptophan fluorescence of GalR at 2.0 ϫ 10Ϫ6 Downloaded by guest on October 7, 2021 Biochemistry: Chatterjee et al. Proc. Natl. Acad. Sci. USA 94 (1997) 2961

the C domain in GalR. A similar change has been documented in ␭ repressor (21). In Vitro Transcription. Because the fluorescence anisotropy and CD results indicated that GalR was bound to the operator in the presence of the inducer, we studied derepression of transcription of the gal P1 promoter in vitro as a function of D-galactose concentration under conditions used for ligand binding studies, except the KCl concentration was 200 mM. The slightly lower salt concentration was chosen to avoid a possible decrease in transcription. The lower salt concentration is expected to further tighten the binding of GalR to the operator. The results are shown in Fig. 7. In the absence of inducer, GalR at 1.6 ϫ 10Ϫ7 M transcription initiation from the P1 promoter repressed by about 70% (Fig. 7A, lane 1 vs. 2). In the presence of 2 ϫ 10Ϫ3 M D-galactose, transcription was derepressed 78%, whereas in the presence of a saturating concentration of the inducer (3 ϫ 10Ϫ2 M) the P1 promoter was derepressed to 99% (lanes 2 and 3). When transcription was studied at 8 ϫ 10Ϫ7 M GalR, the presence of 3 ϫ 10Ϫ2 M D-galactose derepressed transcription by 38% (Fig. 7B). This result is significant considering the fact that at this high GalR FIG. 5. Effect of OE DNA binding on the tryptophan fluorescence concentration, the repressor also inhibited transcription from Ϫ6 (f͞fo) of GalR. GalR at a concentration of 2.0 ϫ 10 M was titrated nonspecific promoters, such inhibition not being lifted by with increasing concentrations of the operator (20-mer). The total D-galactose. Given the determined values of the K s, we emission spectra were integrated. D calculated the fractional occupancy, Y, of operator by repressor from the equation M concentration in the presence of increasing concentrations of OE DNA. As shown in Fig. 5, binding of OE reduced the 1 fluorescence by more than 11%. Y ϭ 1 ϩ KD/Rf [1] Measurements were taken of the distance of the tryptophan residue in GalR from the AEDANS placed at one end of the at 1.6 ϫ 10Ϫ7 M and 8.0 ϫ 10Ϫ7 M GalR. At 2 ϫ 10Ϫ9 M DNA operator DNA. AEDANS has a large overlap in its excitation and such high repressor concentrations, the free repressor spectra with the tryptophan emission spectra. Fig. 6 shows the concentration, Rf, is practically the same as the total repressor Ϫ7 Ϫ7 excitation spectra of the AEDANS-labeled OE and of a concentration. At 1.6 ϫ 10 M and 8.0 ϫ 10 M GalR and stoichiometric complex of AEDANS-OE and GalR. At 295 nm, saturating inducer concentration, transcription is 99% and the fluorescence of the complex was slightly higher than that 38%, respectively, of the unrepressed levels and the calculated of the free repressor, suggesting a small, but significant, energy occupancy of operator by repressor is 86% and 94%, respec- transfer. The calculated energy transfer efficiency was 0.09, tively. which corresponds to a distance of 38.6 Å between the tryptophan and the AEDANS label. Mutational and modeling studies also have placed the tryptophan residue in the C domain and the DNA-binding motif in the N domain of GalR (19, 20). Because the DNA-binding site and the tryptophan residue in GalR are far from each other and in different domains, quenching of tryptophan fluorescence in GalR by operator binding strongly suggested that a DNA-induced conformational change is transmitted from the N domain to

FIG. 6. Fluorescence energy transfer between AEDANS-OE and FIG. 7. The effect of D-galactose on galP1 transcription in the tryptophan of the GalR. The excitation spectra of 20-mer AEDANS- presence of GalR. In vitro transcription assays were carried out as Ϫ6 labeled OE (0.5 ϫ 10 M) (– – – –), and that of the same DNA described in the text. GalR concentrations were 160 (A) and 800 nM complexed with GalR (1.0 ϫ 10Ϫ6 M) (——-) are shown. (B). Downloaded by guest on October 7, 2021 2962 Biochemistry: Chatterjee et al. Proc. Natl. Acad. Sci. USA 94 (1997)

DISCUSSION bound GalR and RNA polymerase brings about repression of the P1 promoter, then binding of inducer to the operator– In negative control, binding of repressor to a cognate operator bound repressor neutralizes the inhibitory contact without inhibits RNA polymerase action at the promoter, and an dissociating the repressor from DNA. A role of repressor at a allosteric modification of repressor by a small-molecule in- post-RNA polymerase binding level also has been suggested in ducer inhibits repressor action. Likely mechanisms by which the E. coli lac promoter and Bacillus subtilis phage ␾ 29 A2c the inducer can inhibit repressor action include: promoter (28–30). As in the gal P1 promoter, MerR repressor (i) Inducer binds only to free repressor, allosterically inac- and RNA polymerase coexist at the repressed promoter in the tivating its operator binding site. In this model, induction mer operon (31, 32). Binding of inducer Hg2ϩ to MerR follows the rate of spontaneous dissociation of operator from derepresses transcription without Hg2ϩ–MerR being dissoci- repressor. ated from the promoter. But unlike the repressor gal P1 (ii) Inducer forms an inducer–repressor–operator ternary complex, the protein-DNA linkage is obligatory for mer tran- complex which, because of allosteric change, dissociates into scription, because the Hg2ϩ–MerR complex is an activator of inducer-repressor complex and free operator more readily the mer promoter. than into repressor–operator complex and free inducer. In this case, induction follows the rate of dissociation of repressor– Note added in proof. Since this manuscript was submitted, we noticed inducer complex from free operator. that the BetI repressor of E. coli binds to each operator both in the (iii) Inducer forms an inducer–repressor–operator ternary absence and presence of inducer (Røkenes, T. P., Lamark, T., Strøm, complex which, because of allosteric change, allows RNA A. R. (1996) J. Bacteriol. 178, 1663–1670). polymerase to act without repressor being physically dissoci- ated from operator. We thank D. Jin and G. Gussin for many critical suggestions and D. GalR binds to operator O and inhibits transcription from Jin also for the gift of plasmid DNA and acknowledge the Council of E Scientific and Industrial Research for a fellowship to S.C. the P1 promoter of the gal operon (6, 22). We showed that D-galactose and the OE operator antagonize the binding of 1. Jacob, F. & Monod, J. (1961) J. Mol. Biol. 3, 318–356. each other to GalR. Consistently, both D-galactose and OE 2. Adhya, S. & Miller, W. (1979) Nature (London) 279, 492–494. DNA binding to GalR exhibit allosteric changes in the protein 3. Irani, M., Orosz, L. & Adhya, S. (1983) Cell 32, 783–788. (ref. 13; results reported in this paper). Despite such an 4. Fritz, H.-J., Bicknase, H., Gleumes, B., Heibach, C., Roshl, S. & antagonistic effect, inducer binding to the operator-bound Ehring, R. (1983) EMBO J. 2, 2129–2135. repressor was found to be only 10-fold less than the extent of 5. Majumdar, A. & Adhya, S. (1984) Proc. Natl. Acad. Sci. USA 81, binding to free repressor under conditions of repression and 6100–6104. induction of gal transcription. Similarly, the affinity of O to 6. Choy, H. E. & Adhya, S. (1992) Proc. Natl. Acad. Sci. USA 89, E 11264–11268. the inducer–repressor complex is reduced by 7-fold compared 7. Buttin, G. (1963) J. Mol. Biol. 7, 164–182. with the binding of OE to the free repressor. The results of 8. Aki, T., Choy, H. E. & Adhya, S. (1996) Genes Cells 1, 179–188. GalR binding to fluorescein-labeled DNA in the presence of 9. Zhou, Y. N., Chatterjee, S., Roy, S. & Adhya, S. (1995) J. Mol. D-galactose also showed the existence of stoichiometric Biol. 253, 414–425. amounts of inducer–repressor–operator ternary complex. The 10. Wartell, R. & Adhya, S. (1988) Nucleic Acids Res. 16, 11531– existence of stable ternary complex under physiological con- 11541. ditions also has been confirmed by comparing CD spectra of 11. Cantor, C. & Schimmel, P. (1981) Biophysical Chemistry (Free- the free operator, operator bound to repressor, and operator man, San Francisco), Vol. 2. bound to repressor in the presence of inducer. Comparable 12. Wu, C. W. & Stryer, L. (1972) Proc. Natl. Acad. Sci. USA 69, 1104–1108. reduction in the affinity of inducer for repressor when com- 13. Brown, M., Shaikh, N., Brenowitz, M. & Brand, L. (1994) J. Biol. plexed with operator was found in the lac system (23, 24). Chem. 269, 12600–12605. In vitro transcription results presented here clearly demon- 14. Nakanishi, S., Adhya, S., Gottesman, S. & Pastan, I. (1973) Proc. strated that full derepression of gal P1 occurs under conditions Natl. Acad. Sci. USA 70, 334–336. of binding in which (i) the repressor is still bound to the DNA, 15. Letilly, V. & Royer, C. A. (1993) Biochemistry 33, 7753–7758. i.e., the concentrations of inducer–repressor binary complex 16. Brenowitz, M., Jamison, E., Majumdar, A. & Adhya, S. (1990) are severalfold higher than the dissociation constant of the Biochemistry 29, 3374–3383. inducer–repressor–operator ternary complex into the inducer- 17. Ha, J. H., Capp, M. W., Hohenwalter, M. D., Baskerville, M. & Record, M. T., Jr. (1992) J. Mol. Biol. 228, 252–264. –repressor complex and free DNA; (ii) the RNA polymerase 18. Zwieb, C., Kim, J. & Adhya, S. (1989) Genes Dev. 3, 606–611. concentration was significantly less than the dissociation con- Ϫ1 19. Lehming, N., Sartorius, J., Kister-Woike, B., von Wilcken-Berg- stant of the RNA polymerase-promoter complex (KB ) (25, 26), mann, B. & Muller-Hill, B. (1990) EMBO J. 9, 615–621. and (iii) nonspecific DNA concentration was negligible. These 20. Hsieh, M., Hensley, P., Brenowitz, M. & Fetro, J. S. (1994) J. Biol. results strongly suggest that previous dissociation of repressor Chem. 269, 13825–13835. is not obligatory for full derepression of transcription from the 21. Saha, R., Banik, U., Bandopadhyay, S., Mandal, N. C., Bhatta- P1 promoter of the gal operon. Whether gal derepression charyya, B. & Roy, S. (1992) J. Biol. Chem. 267, 5862–5867. occurs by a similar mechanism in vivo remains to be investi- 22. Choy, H. E., Park, S.-W., Parrack, P., Fujita, N., Ishihama, A. & Adhya, S. (1995) EMBO J. 14, 4523–4529. gated. 23. Chen, J., Alberti, S. & Matthews, K. S. (1994) J. Biol. Chem. 269, It is unclear how repressor inhibits transcription. Operator- 12482–12487. bound repressor either occludes RNA polymerase binding to 24. Daly, T. J. & Matthews, K. S. (1986) Biochemistry 25, 5479–5484. the promoter or inhibits RNA polymerase activity at one or 25. Goodrich, J. A. & McClure, W. R. (1992) J. Mol. Biol. 224, 15–29. more of the postbinding steps (27). We previously demon- 26. Herbert, M., Kolb, A. & Buc, H. (1986) Proc. Natl. Acad. Sci. USA strated that GalR and RNA polymerase coexist on the DNA, 83, 2807–2811. and GalR inhibits the P1 promoter by freezing the RNA 27. Adhya, S. (1989) Annu. Rev. Genet. 23, 227–250. polymerase at an intermediate step between closed and open 28. Straney, S. B. & Crothers, D. M. (1987) Cell 51, 699–707. 29. Lee, J. & Goldfarb, A. (1991) Cell 66, 793–798. complex formation (22). GalR does so by modulating the 30. Monsalve, M., Menica, M., Salas, M. & Rojo, F. (1996) Proc. Natl. activity of the ␣ subunit of the DNA-bound RNA polymerase. Acad. Sci. USA 93, 8913–8918. As discussed above, dissociation of repressor from the oper- 31. Summers, A. O. (1992) J. Bacteriol. 174, 3097–3101. ator is not obligatory for transcription from the P1 promoter. 32. Ansari, A. Z., Bradner, J. E. & O’Halloran, T. V. (1995) Nature We propose that because a direct contact between DNA- (London) 374, 371–375. Downloaded by guest on October 7, 2021